Function And Mechanism Of Scorpion Toxin BmKM9 In Inhibiting Hnav1.5 Sodium Channel In Breast Cancer

Objective:To study expression of hNav1.5 sodium channels in breast cancer cell of different subtypes,and to explore correlation between expressive level of hNav1.5 and the severity of the tumor;As a result,Biological Function and Mechanism that peptide-BmKM9 Specifically inhibitor Nav1.5 sodium channels will be illuminated in breast cancer cell and experimental rats models,establish the foundation for the prevention and treatment of breast cancer.Methods:1.The sequence of BmKM9 was cloned into a prokaryotic vector,and use X1X5X4BH-28a-BL21(DE)3 induce protein expression.Then,the protein was purified by nickel column and high performance liquid chromatography(HPLC),and the protein fusion head was removed by rTEV enzyme to obtain highly purified recombinant BmKM9.2.We can extract proteins from different subtypes of the human breast cancer cell line,and use western blot to analyze hNav1.5 content in different samples.Select the cell lines with high expression according to the expression level of hNav1.5.3.Methods Whole cell patch clamp technique was used to record effects of BmKM9 on the sodium current in breast cancer cell MDA-MB-231.4.BmKM9 was co-incubated with breast cancer cells,and the effects of BmKM9 on breast cancer cell proliferation,migration,invasion,apoptosis,and cell cycle were detected by MTT method,scratch method,invasion test,PI/FITC.5.The cultured breast cancer cells added into BmKM9,used the qPCR array to quantitatively analyze protein expression changes in the signal pathway by BmKM9 affected,compared the differences of the mRNA expression among control group and BmKM9 group.According to these differentially expressed genes,relevant protein antibodies corresponding to the signaling pathways are selected and verified again at the protein level to identify the main signaling pathways for BmKM9 to play biological functions.6.we established nude mice transplantable mammary tumor model.Fifteen nude mice were divided randomly into three groups,5 rats in each groups.the control group(control group,saline injection)and ranolazine group(50 mg/kg)(positive control),BmKM9(lmg/kg)group,continuous administration for 1 month,complete tumor stripping,and use the immunohistochemistry(IHC),immunofluorescence to detect K167 level in tumor tissues.Results:1.Western blot results showed that hNavl.5 was not expressed or expressed at low levels in normal breast cells and weakly invasive breast cancer cells,but in triple-negative breast cancer cells(TNBC)with strong invasive capabilities such as:MDA-MB-231 presents high expression.2.The electrophysiological activity of hNav1.5 sodium channel in MDA-MB-231 cells was greatly suppressed by 1?M BmKM9,while 0.0625?M-2?M BmKM9 has no obvious promotion or inhibition effect on breast cell/breast tumor cell proliferation.3.The breast cancer cells treated with 1?M BmKM9 had significantly reduced migration and invasion capabilities.4.BmKM9 had no effect on cell cycle and apoptosis.5.Quantitative analysis of qPCR array show that expression of some genes in MDA-MB-231 cell line was significantly up or down regulated in BmKM9 group compared with control group.These differentially-expressed genes mostly fund in wnt pathway after statistical analysis of the data.Some antibodies in the WNT pathway were used to verify related proteins again,and it was found that the expression of?-catanin protein in MDA-MB-231 cells treated with BmKM9 was significantly down-regulated.6.In animal models of orthotopic breast cancer transplantation,the control(saline injection)group,the Ranolazine(50mg/kg)group,and the BmKM9(1mg/kg)group were observed and recorded for one month.The tumor volume in the Control group was smaller than the Ranolazine group,The BmKM9(1 mg/kg)group was large,and tumor weight and volume were consistent.IHC results of tumor samples showed that KI67 levels in Ranolazine group and BmKM9(lmg/kg)group were signiificantly lower than those in Control group.Conclusion:The hNav1.5 high expression in breast cancer cells with high malignancy and aggressiveness,which is related to the prognosis of breast cancer.The small peptide-BmKM9 isolated and purified from scorpion toxin can specifically inhibit the hNav1.5 ion channel and reduce the invasion and migration ability of breast cancer cells.Injecting BmKM9 in mice can inhibit the growth of breast cancer tumors;BmKM9 can down-regulate ?-catenin to inhibit the activation of Wnt/?-catenin signaling pathway,thereby reducing the metastatic capacity of breast cancer cells.As for how BmKM9 affects the WNT/?-catenin pathway by inhibiting the electrical activity of hNav1.5,the mechanism of action is still unclear and needs to be further explored.